Introduction

TOPO Tools technology uses the unique properties of DNA topoisomerase I to allow rapid, directional topoisomerase I-mediated joining (“TOPO Joining”) of PCR products to a choice of 5′ and 3′ elements. You will choose the 5′ and 3′ elements depending on the application you wish to perform. Once your PCR product and the 5′ and 3′ elements are TOPO Joined, the resulting linear DNA template is amplified using the element specific primers to generate the linear DNA construct. This linear DNA construct can be used directly in the appropriate application without the need for additional cloning or transformation steps.

TOPO Tools Elements for in vitro Transcription /Translation

The TOPO Tools Elements for in vitro Transcription/Translation are designed for directional topoisomerase I mediated joining of your PCR products to a choice of 5′ or 3′ element to create a linear DNA construct. The resulting linear DNA construct is used to generate RNA transcripts or protein from your PCR product. The important features of the TOPO Tools Elements for in vitro Transcription/ Translation are listed below:

T7 or T3 promoter sequences for transcription of your PCR product using the T7 or T3 RNA polymerase

Topoisomerase binding site (CCCTT) for directional topoisomerase I-mediated joining of your PCR product to the 5′ or 3′ element of choice

Polyadenylation (pA) sequence for stabilization of mRNA transcripts

Priming site for secondary amplification of the linear DNA template to generate a linear DNA construct

Depending on the desired downstream application, seven types of TOPO Tools Elements for in vitro Transcription/Translation Kits are available to allow you to generate a linear DNA construct of your choice

How to follow this page

To create a linear DNA construct and use the linear DNA construct for appropriate downstream applications such as in vitro transcription or coupled in vitro transcription/translation, you will need instructions from this webpage and the TOPO Tools Technology manual.

This webpage provides the following information:

Sequences of the 5′ and 3′ TOPO-adapted elements

Designing PCR primers

Guidelines to perform in vitro transcription or coupled in vitro transcription/translation

For information on the TOPO Tools technology, please refer to the TOPO Tools Technology manual. You may download the manual from our web site at www.thermofisher.com/cloning or contact Technical Service. The TOPO Tools manual includes the information to:

Perform the control reaction using the CAT control PCR templateControl Reaction

Using an Appropriate Element

Seven types of elements are available to create a linear DNA construct of your choice. Use the appropriate element based on your specific application.

T7 5' or T3 5' Element for in vitro transcription of your gene of interest in the sense orientation using the T7 or T3 RNA polymerase.

T7 3' or T3 3' Element for in vitro transcription of your gene of interest in the anti-sense orientation using the T7 or T3 RNA polymerase.

T7 Kozak 5' Element and the pA 3' Element for coupled in vitro transcription/translation of your gene of interest using the T7 RNA polymerase. This element contains the Kozak consensus sequence for optimal translation of your gene of interest.

Note: If your gene of interest contains an ATG with a Kozak consensus sequence, you may use the T7 5′ or T3 5′ Element and the pA 3′ Element for coupled in vitro transcription/translation of your gene of interest.

Experimental Outline

Introduction

The table below outlines the experimental steps necessary to perform in vitro transcription or coupled in vitro transcription/translation using TOPO Tools T7 or T3 elements and your gene of interest. Please refer to the indicated manual for more details on each step. We recommend that you read the entire TOPO Tools Technology manual to familiarize yourself with the technology and the various steps necessary to generate a linear DNA construct containing your gene of interest and a choice of TOPO-adapted element.

Amplify your linear DNA template with a thermostable polymerase to generate a linear DNA construct using the element specific primers included in the kit.

5

Verify the integrity and estimate the concentration of your PCR product.

6

Use the linear DNA construct in the downstream application of choice:

In vitro transcription

Coupled in vitro transcription/translation

TOPO Tools Elements for in vitro Transcription/Translation

High-Throughput Application

If you need to perform the TOPO Tools technology in a high-throughput format, please refer to the TOPO Tools Technology manual for more guidelines. You can amplify your gene of interest, perform the TOPO Joining reaction, secondary amplification reaction, and in vitro transcription or translation in a high-throughput format.

A six base pair sequence which base pairs with the overhang sequence on the TOPO-adapted 5′ or 3′ element. The six base pair sequence facilitates directional TOPO Joining and differs for the forward and reverse primer (see below)

A five base pair sequence (AAGGG, shown in bold in the sequences below) which is complementary to the topoisomerase I recognition site (CCCTT) to increase the efficiency of topoisomerase I-mediated joining of the PCR product to the 5′ and 3′ elements

Forward Primer: 5′-CGGAACAAGGG-3′ Reverse Primer: 5′-TGAGTCAAGGG-3′

For more details and an example of designing PCR primers for proper TOPO Joining and generation of the linear DNA construct, please refer to the TOPO Tools Technology manual.

To obtain consistent and efficient results in the TOPO Joining reaction, we highly recommend that you use HPLC-purified oligonucleotides to produce your PCR products. Using a mixture of full-length and non full-length primers to produce your PCR products can reduce the efficiency of TOPO Joining and result in poor yield of linear DNA construct after secondary amplification. Once you have obtained your HPLC-purified oligonucleotides, we also recommend that you use polyacrylamide gel electrophoresis to verify the length of your oligonucleotides.

Do not add 5´ phosphates to your primers for PCR. This will prevent TOPO Joining.

Be sure to include a stop codon in the reverse primer or design the reverse primer to hybridize downstream of the native stop codon, if you wish to translate your gene of interest.

In vitro Transcription and Coupled in vitro Transcription/Translation

Introduction

Once you have generated a linear DNA construct containing your gene of interest and a TOPO Tools element of choice, you are ready to use the linear DNA construct for your downstream application such as in vitro transcriptio or coupled in vitro transcription/translation. You may use the protocol of choice or commercially available kits to perform in vitro transcription or coupled in vitro transcription/translation reaction. If you are using commercially available kits, please refer to the manufacturer’s recommendations.

For general information and protocols on in vitro transcription or coupled in vitro transcription/translation, please refer to Current Protocols in Molecular Biology (Ausubel et al., 1994).

Before Starting

Before performing in vitro transcription or coupled in vitro transcription/translation, be sure to have the following:

100-200 ng of your linear DNA construct that you created by TOPO Joining the PCR product with a choice of T7 or T3 Elements.

A single discrete band corresponding in size to the linked linear DNA construct of sufficient yield after the secondary amplification. If you need to purify the PCR product, you may use the protocol provided in the Appendix section of the TOPO Tools Technology manual. Other protocols are suitable.

General Handling of RNA

Please note that the transcription reaction produces RNA. When working with RNA, be sure to follow the tips listed below to prevent RNA degradation:

Use disposable, individually wrapped, sterile plasticware.

Use only sterile, new pipette tips and microcentrifuge tubes.

Wear latex gloves while handling reagents and RNA samples to prevent RNase contamination from the surface of the skin.